Regulatory T cells (Treg or Tregs) have become a major focus of immunological research due to their role as interface between establishing tolerance against harmless self and foreign antigens on one side and allowing counteraction against harmful antigens on the other side. Increased levels of Treg have been found in many human cancers. This has promoted the theory that dysregulation of Treg levels leads to overzealous tolerance against tumor cells. The Treg property to dampen immune reactions is also important for therapeutic strategies involving modulation of the immune system.
The transcription factor Foxp3 is specifically expressed in regulatory T cells and is thought to function as a master switch for the development and function of these cells. Recently, it has been demonstrated that ectopic expression of Foxp3 in conventional T cells confers suppressive activity (Fontenot and Rudensky, Nat Immunol 6:331-337, 2005).
The vast majority of Foxp3+ regulatory T cells is generated during T cell development within the thymus, and it is thought that they represent an individual lineage. In addition, it also has been reported that Foxp3+ regulatory T cells arise from conventional T cells both in vitro and in vivo upon antigen recognition under tolerogenic conditions. In all cases the expression of Foxp3 is characteristic for the development of regulatory T cells.
It is largely unknown, which signals lead to the expression of Foxp3, although some factors including TGF-β have been reported to induce Foxp3 expression in conventional T cells. However, it is unknown if TGF-β induction leads to a full differentiation into a Treg, or merely to an only transiently FOXP3 expressing T cells, with or without a suppressive phenotype. Therefore, the starting point for the present invention was the search for phenotypes clearly related to T cells having a stable suppressive phenotype.
One obstacle for monitoring Treg levels is that the best current detection method, the analysis of FOXP3 and CD25 mRNA and/or protein, detects both, Tregs and activated T-cells, and is therefore unable to distinguish between these phenotypes.
Even though almost all cells in an individual contain the exact same complement of DNA code, higher organisms must impose and maintain different patterns of gene expression in the various tissue types. Most gene regulation is transitory, depending on the current state of the cell and changes in external stimuli. Persistent regulation, on the other hand, is a primary role of epigenetics—heritable regulatory patterns that do not alter the basic genetic coding of the DNA. DNA methylation is the archetypical form of epigenetic regulation; it serves as the stable memory for cells and performs a crucial role in maintaining the long-term identity of various cell types.
The primary target of methylation is the two-nucleotide sequence Cytosine-Guanine (a ‘CpG site’); within this context cytosine (C) can undergo a simple chemical modification to become 5-methyl-cytosine. In the human genome, the CG sequence is much rarer than expected except in certain relatively dense clusters called ‘CpG islands’. CpG islands are frequently associated with gene promoters, and it has been estimated that more than half of the human genes have CpG islands (Antequera and Bird, Proc Natl Acad Sci USA. 90:11995-9, 1993).
Aberrant methylation of DNA frequently accompanies the transformation from healthy to cancerous cells. Among the observed effects are genome-wide hypomethylation, increased methylation of tumor suppressor genes and hypomethylation of many oncogenes (reviewed by Jones and Laird, Nature Genetics 21:163-167, 1999; Esteller, Oncogene 21:5427-5440, 2002; Laird, Nature Reviews/Cancer 3:253-266, 2003). Methylation profiles have been recognized to be tumor specific (i.e., changes in the methylation pattern of particular genes or even individual CpGs are diagnostic of particular tumor types) and there is now an extensive collection of diagnostic markers for bladder, breast, colon, esophagus, stomach, liver, lung, and prostate cancers (summarized by Laird, Nature Reviews/Cancer 3:253-266, 2003).
Chen et al. (Chen L, Cohen A C, Lewis D B. Impaired Allogeneic Activation and T-helper 1 Differentiation of Human Cord Blood Naive CD4 T Cells. Biol Blood Marrow Transplant. 2006 February; 12(2):160-71) describe FoxP3 protein expression as a marker for regulatory CD25(high) CD4 T cells.
EP 1213360 describes a method of identifying a cell, tissue or nucleus, comprising collecting information on the methylation pattern of DNA isolated from the cell, tissue or nucleus and analyzing the resultant information.
WO 2004/050706 describes a sub-group of T-cells, and relates to characteristics of regulatory T-cells which define them as such. The application also describes the uses of such T-cells, compositions comprising them and chemokines which recruit them in the modulation of an immune response.
Finally, EP 1826279 describes a method, in particular an in vitro method for identifying FoxP3-positive regulatory T cells, preferably CD25+ CD4+ regulatory T cells of a mammal, comprising analyzing the methylation status of at least one CpG position in the gene foxp3 or an orthologous or paralogous gene thereof, and the use of DNA-methylation analysis of the gene of the transcription factor FoxP3 for a detection and quality assurance and control of regulatory T cells.
In view of the above, it is an object of the present invention, to provide an improved method based on DNA methylation analysis as a superior tool in order to more conveniently and reliably identify FoxP3-positive stable regulatory T cells, preferably CD25+CD4+ regulatory T cells, derived from a mammal, and/or in a mammal. Furthermore, reliable detection of the phenotype should be available independently of purity, storage, and to some extent also of tissue quality.